Abnormal cellular proliferation, a hallmark of cancer, can result from a wide range of cellular phenomena. Proliferative signals are transmitted into and within a cell via a process known as signal transduction. Over the past decades, cascades of signal transduction pathways have been elucidated and found to play a central role in a variety of biological responses. Defects in various components of signal transduction pathways have been found to account for a vast number of diseases, including numerous forms of cancer, inflammatory disorders, metabolic disorders, vascular and neuronal diseases (Gaestel et al. Current Medicinal Chemistry (2007) 14:2214-2234).
Kinases constitute a large family of important signaling molecules. Kinases can generally be classified into protein kinases and lipid kinases, and certain kinases exhibit dual specificities. Protein kinases are enzymes that phosphorylate other proteins and/or themselves (i.e., autophosphorylation). Protein kinases can be generally classified into three major groups based upon their substrate utilization: tyrosine kinases which predominantly phosphorylate substrates on tyrosine residues (e.g., erb2, PDGF receptor, EGF receptor, VEGF receptor, src, abl), serine/threonine kinases which predominantly phosphorylate substrates on serine and/or threonine residues (e.g., mTorC1, mTorC2, ATM, ATR, DNA-PK), and dual-specificity kinases which phosphorylate substrates on tyrosine, serine and/or threonine residues.
The mammalian target of rapamycin (mTor) is a serine-threonine kinase related to the lipid kinases of the phosphatidylinositol 3 kinase (PI3K) family. mTor has been implicated in a wide range of biological processes including cell growth/proliferation, cell motility and survival. Dysregulation of the mTor pathway has been reported in various types of cancer. mTor is a multifunctional kinase that integrates growth factor and nutrient signals to regulate protein translation, nutrient uptake, autophagy and mitochondrial function.
mTor exists in two complexes, mTorC1 and mTorC2. mTorC1 contains the raptor subunit and mTorC2 contains rictor. These complexes are differentially regulated, and have distinct substrate specificities and rapamycin sensitivity. For example, mTorC1 phosphorylates S6 kinase (S6K) and 4EBP1 (eIF4E-binding protein 1, also known as also known as EIF4EBP1), promoting increased translation and ribosome biogenesis to facilitate cell growth and cell cycle progression. S6K also acts in a feedback pathway to attenuate PI3K/Akt activation. mTorC2 is generally insensitive to rapamycin. mTorC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt. In many cellular contexts, mTorC2 is required for phosphorylation of the S473 site of Akt.
The serine/threonine kinase Akt (also known as protein kinase B) possesses a pleckstrin homology (PH) domain that binds PIP3, leading to Akt kinase activation. Akt phosphorylates many substrates and is a central downstream effector of PI3K for diverse cellular responses (FIG. 1). Full activation of Akt typically requires phosphorylation of T308 in the activation loop and S473 in a hydrophobic motif. One important function of Akt is to augment the activity of mTor, through phosphorylation of TSC2 and other mechanisms.
Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinase enzymes whose products mediate reversible membrane localization of cytoplasmic proteins. PI3K activation in most cells correlates with proliferation and suppression of apoptosis. PI3K and its downstream effectors also control cell polarity, motility, metabolism and other physiological processes. This signaling pathway also plays a prominent role in cancer: the PI3K pathway activity is enhanced in nearly all human tumors. This can occur by gain-of-function mutations or amplifications of PI3K genes, loss of the tumor suppressor PTEN (the major lipid phosphatase that opposes PI3K signaling), or expression of oncogenes that activate PI3K (FIG. 1). In mouse models, enhanced PI3K signaling in lymphocytes leads to lymphoproliferation, susceptibility to leukemia, and spontaneous autoimmunity. Conversely, deletion of PI3K genes causes immunodeficiency and resistance to malignant transformation. Pharmacological suppression of immune responses and cancer cell proliferation can also be achieved using rapamycin, which inhibits mTor (mammalian target of rapamycin) downstream of PI3K (FIG. 1).
The PI3K/Akt/mTor signaling axis has been extensively studied with small molecule inhibitors. Wortmannin and LY294002 are two broad-spectrum PI3K inhibitors that have potent anti-proliferative effects; however, these agents have broad inhibition activity towards most PI3K isozymes as well as other cellular targets. A key example of these off-target effects is the direct inhibition of mTor by LY294002 (and wortmannin at high concentrations).
Conventional mTor selective inhibitors also suffer from several profound drawbacks. For example, the mTor inhibitors, namely rapamycin and analogs, also termed “rapalogs” are potent immunosuppressants. They have also been used in clinical trials for various types of cancer. Unfortunately, the results of these clinical trials to date have been mixed, with few malignancies showing consistent response to rapalogs. Rapamycin (RAP) has a mechanistic limitation: it is an allosteric, noncompetitive inhibitor of mTorC1 that does not acutely inhibit mTorC2 in most cells. Hence, cells treated with RAP usually display increased Akt phosphorylation on both T308 and S473, due to loss of the feedback inhibitory circuit mediated by S6K (FIG. 1). This can lead to chemoresistance of cancer cells treated with rapalogs. Although RAP does inhibit mTorC2 in some cell types by disrupting assembly of the complex, the phenomenon of rapamycin-induced stimulation of Akt has been observed in many settings. It is also worth noting that mTorC2 might have additional functions in tumor cells, other than Akt-S473 phosphorylation, which remain unaffected by RAP.